DNA damage is the most common cause of cancer. In response to DNA damage, mammalian cells arrest cell cycle progression by signal transduction mechanisms called DNA damage checkpoints, repair the DNA, and resume the cell cycle. Defects in DNA damage checkpoints or repair mechanisms cause genomic instability and cancer. The goal of our research is to understand the molecular mechanisms of DNA excision repair and the DNA damage checkpoints. We will perform biochemical experiments to characterize these pathways. I. DNA EXCISION REPAIR. This repair system removes DNA damage by dual incisions bracketing the lesion. It repairs all base lesions, and it is the sole repair system for bulky DNA adducts. Excision repair has been reconstituted in vitro and characterized in some detail; but the mechanism by which it recognizes the most common carcinogenic DNA lesion, the cyclobutane thymine dimer, is not known. We will determine how the thymine dimer and other lesions are recognized by this repair system, how the 6 repair factors of the excision nuclease assemble at the damage site and disassemble following dual incisions, and how this repair system deals with DNA protein crosslinks that are often caused by anticancer drugs. II. BIOCHEMICAL PROPERTIES OF DNA DAMAGE CHECKPOINT PROTEINS. Checkpoint proteins include damage sensors, mediators, signal tranducers, and effectors. We will characterize the damage sensors ATR, Rad17-RFC, and the 9-1-1 complex with respect to their interactions with damaged DNA and with the other components of the checkpoint pathway. We will purify the mediators, claspin, MDC1, and Rad5, and analyze their interactions with DNA, checkpoint sensors, and checkpoint kinases. III. DNA DAMAGE CHECKPOINT IN VITRO. Currently there is no in vitro system for studying the mammalian DNA damage checkpoints. We will develop in vitro systems based on permeabilized nuclei and nuclear extracts for studying the initial steps of the checkpoint response, including damage-recognition, recruitment of mediators to the assembly site, and activation of the checkpoint kinases. These studies will provide biochemical tests of current checkpoint models that are based on genetic and cellular analyses.